Introduction

The purpose of this tutorial is to provide a step-by-step guide to isolate nuclei from flash-frozen Kidney synthetic assembloids for single-nucleus RNA sequencing. Nuclei Isolation is the first step in many novel techniques including snNRAseq, single-nucleus assay for transposase-accessible chromatin sequencing, epigenomic analysis. Although based off of the 10X Genetics Chromium Nuclei Isolation Protocol, this tutorial is specifically for the use of Kidney synthetic assembloids for snRNAseq and includes alterations to maximize yield and quality. This tutorial will also include a guide on how to assess and grade the quality of the isolated nuclei as well as next steps after the initial isolation.

Overview of Procedure

Link to 10X Protocol, starts on Page 25

Video Walk-Though of Procedure

Future Link

PDF Download of Protocol

Link to Printable PDF of Protocol

Tips

Critical to pellet the Assembloids prior to flash freezing in the sample tubes supplied by 10X chromium so it fits the pestle.

Work quickly helps with maintaining the quality of the nuclei.

Depending on the starting material and step that your working on, it might be challenging to visualize the nuclei (especially after the debris removal step), so make sure to know the orientation of the tubes in the centrifuge.

Waiting 15 minutes at the end of the protoco help for the damaged nuclei to clump together so that mostly high-quality nuclei remain in the sample and are resuspended.

Key Modifications

Multiple modifications have been made to optimize this protocol for the small tissue of assembloids.

Decreased volume of Lysis Buffer needed (100 µL)

Decreased number of pestle twists to dissociate tissue

We found that 5 was suffecient but **vissually confirmed the solution was absense of large chunks

Skip the step to run solution through a column to remove large debris

Perform ALL centrifugations in a Round Bottom FACS tube and a Swinging Bucket Rotor

Decreased Wash Buffer volumes to 500 µL

Final resuspension in 100 µL

Consumables

List of Consumables
Action Item 10X PN Preparation & Handling Storage
Place on Ice Lysis Reagent 2000558 Vortex, verify no precipitate, and centrifuge briefly. 4°C
Surfactant A 2000559 Vortex, verify no precipitate and centrifuge briefly. 4°C
Debris Removal Reagent 2000560 Vortex, verify no precipitate and centrifuge briefly. 4°C
Reducing Agent B 2000087 Thaw to RT, vortex, verify no precipitate, and centrifuge briefly. −20°C
RNase Inhibitor 2000565 Centrifuge briefly. −20°C
Nuclei Isolation Consumables:
Nuclei Isolation Column 2000562 Pre-chill both on ice Ambient
Collection Tube 2000563 Pre-chill both on ice Ambient
Nuclease-free Water — See Buffer Preparation Ambient
1X PBS — See Buffer Preparation Ambient
10% BSA — See Buffer Preparation 4°C
Round Bottom FACS tubes Corning (352003) Ambient
Place on Dry Ice Frozen Tissue Sample — Thaw on ice Liquid Nitrogen (long-term) or −80°C (short-term)
Sample Dissociation Tube 2000564 Pre-chill on dry ice Ambient
Obtain Pestles 2000561 Keep on lab bench Ambient
Nucleic Acid Staining Fluorescent Dye — See Tips & Best Practices 4°C
Vortex — See Nuclei Isolation Protocol —

Equipment

Eppendorf 5810R Refrigerated Centrifuge

Link to product page.

Buffer Set-Up

Buffer Setup
Buffer Name Component 1 Component 2 Compound 3
1X PBS 10 mL of 10X PBS 90 mL DI water N/A
10% BSA 10 g BSA powder (4°C) 100 mL DI water N/A
Lysis Buffer Lysis Reagent (4°C) Surfactant A (4°C) Reducing Agent B (-20°C)
Debris Buffer Debris Removal Reagent (4°C) Reducing Agent B (-20°C) N/A
Wash and Resuspension Buffer 1X PBS 10% BSA (4°C) Rnase Inhbitor (-20°C)

1. Start with the Wash and Resuspension Buffer, which is composed of the 1X PBS and 10% BSA solutions that are not included in the kit. Generally, make a stock of 100 mL of each and keep chilled at 4°C. Refer to the table above and the instructions for components and amounts.

1X PBS: Dilute 10 mL into 90 mL of Ultra-Pure Water. Invert to mix thoroughly.
10% BSA: 10 grams of powder BSA to 100 mL of Ultra-Pure Water. Mix gently by swirling to prevent excess frothing.

2. Next, retrieve all components of the 10X Nuclei Isolation Kit, which should include the Lysis Reagent, Debris Removal Reagent, Reducing Agent B, Surfactant A, and RNAse Inhibitor. Also retrieve the Sample Dissociation Tube from the room temperature Consumables portion.

3. Place everything except for the Reducing Agent B on ice and begin to prepare the buffers. After Reducing Agent B thaws and everything is briefly vortexed, begin preparing the buffers.

4. Make an appropriate amount of buffer for the number of samples that you are going to be isolating nuclei from

Lysis Buffer
Checkbox Solution PN 1X+10% (µl) 4X+10% (µl) 8X+10% (µl)
â–¡ Lysis Reagent 2000558 550.00 2200.0 4400.0
â–¡ Reducing Agent B 2000087 0.55 2.2 4.4
â–¡ Surfactant A 2000559 5.50 22.0 44.0
Total 556.05 2224.2 4448.4
Debris Removal Buffer
Checkbox Solution PN 1X+10% (µl) 4X+10% (µl) 8X+10% (µl)
â–¡ Debris Removal Reagent 2000560 550.00 2200.0 4400.0
â–¡ Reducing Agent B 2000087 0.55 2.2 4.4
â–¡ Total NA 550.55 2202.2 4404.4
Wash and Resuspension Buffer
Checkbox Solution PN 1X+10% (µl) 4X+10% (µl) 8X+10% (µl)
â–¡ 1X PBS (not provided) NA 2887.5 11550 23100
â–¡ 10% BSA (not provided) NA 330.0 1320 2640
â–¡ RNase Inhibitor 2000565 82.5 330 660
Total NA 3300.0 13200 26400

5. Keep all completed buffers labeled and on ice. Pre-chill the centrifuge to 4°C.

Main Procedure

1. Retrieve Kidney Synthetic Assembloids from the -80°C storage and place on ice and let it thaw.

2. Add 100 µL of the Lysis Buffer as soon as the tube is thawed.

3. Then using the green pestle provided in the room-temperature pack, grind up the tissue, pressing down and twisting 5 times (avoiding the generation of bubbles) or until the solution is smooth with no visible chunks.

4. Next, add 100 µL of the Lysis buffer and pipette up and down, making sure that there are no chunks.

5. Pre-coat round bottom FACS tubes by pipetting 100 of 10% BSA into tube, swirl around and then discard. Transfer the nuclei solution into the round bottom FACS tube.

6. Spin the sample in a swinging bucket centrifuge at 500 rcf for 3 min at 4°C.

7. Next, remove the supernatant, being careful to avoid the pellet. OK to leave a minimal volume of solution in the tube (~100 µL).

8. Re-suspend the pellet with 500 µL of the Debris Removal Buffer. Spin at 700 rcf for 10 minutes at 4°C.

9. Remove the supernatant again and resuspend in 500 µL of the Wash and Resuspension Buffer. Spin at 500 rcf for 5 minutes, 4 °C. Remove supernatant.

10. Repeat step 9.

11. Finally, resuspend the. nuclei pellet in 100 µL of the Final Wash and Resuspension Buffer. Vortex the solution for 3 seconds and let it sit undisturbed for 15 minutes.

Now, depending on QC or following uses, use the concentrated solution.

Expected Outcomes

In our hands, we started with ~400K nuclei from 5 Mature Kidney Synthetic Assembloids (quantified after initial lysis)

After completing this protocol, we ended up with ~250K nuclei in 100 µL at a concentration of 2,025 nuc/µL

This means that the each quadrant on the hemocytometer had approcimately 20 healthy nuclei

Nuclei Count with Light Microscope:

With the nuclei solution, add 2 µL of nuclei to 18 µL of Trypan Blue, pipetting up and down into the TB at least 10 times to mix thoroughly. Note that while Trypan Blue does not accumulate in intact cells, the compound does concentrate within the exposed nuclei, which is what we will be checking for.

Add 10 µL to one of the channels on a hemocytometer, making sure the solution fills the space. Observe on a light microscope, counting to get an approximation and assessment of nuclei quality.

Microscope used in this procedure:

link to hemocytometer counter app

From this procedure, the average expected nuclei concentration count is: 500 nuclei per microliter This is approximately 20 to 40 nuclei per quadrant in the hemocytometer.

Nuclei Quality Check:

Priority Check:

  1. No clumps, with an even distribution

  2. Quality:

A grade Nuclei: Good, circular shape with no blebbing, halo of Trypan Blue around the edges that suggest an undisturbed nuclear envelope. No aggregation.

B grade Nuclei: Slightly irregular shapes, still has the halo but less bright or more uneven

C grade Nuclei: Losing more of normal shape, small blebbing, paler color

D/F grade Nuclei: Complete loss of structure, no blue halo, aggregates

Example of a graded sample:

Example of grading with reasoning. Note: this is an image of an aggregate that was included because of a wide variety of quality, but as a general rule aggregates indicate a low quality overall sample.

Example of a sample viewed at 10x and 40x, grading the individual nuclei quality is easier at 40x but it is recommended to conduct overall sample quality checks at 10x magnification.